The major goals of this proposal are to establish a series of immortalized cultures from normal, smoke damaged and premalignant bronchial epithelia that retain their genetic and morphologic characteristics, to characterize the cells for their DNA, RNA and protein profiles, to utilize these tools to identify genes that are both up and down regulated during multistage pathogenesis, and to use our findings to identify and validate markers for early lung cancer diagnosis and risk assessment. In Aim 1 we will utilize a novel methodology to immortalize bronchial epithelial cells without the use of oncoproteins. We will immortalize about 20 cultures from never, former and current smokers and smokers with cancer. Some will be paired with corresponding tumor cultures. Lymphoblastoid cultures will be established as sources of constitutional DNA. In Aim 2 we will fully characterize our cells using conventional, novel and high throughput technologies, including identification of the differentiation/premalignant state of the cells using organotypic and subrenal capsule assays, microarrays for gene expression and a very high density array comparative genomic hybridization methodology to identify up and down regulated genes. We will use genetic immunization to produce antibodies for detection of over expressed genes and develop methylation assays for under expressed genes. In aim 4 we will validate these assays in tumors, tissues and sera of patients at increased risk and with lung cancer. This project represents a collaborative effort of scientists and clinicians at UT Southwestern Medical Center, MD Anderson Cancer Center, and the British Columbia Cancer Agency. We will utilize the considerable resources gathered by our Texas SPORE in Lung Cancer grant. We will freely share our reagents and data with other EDRN scientists. Our proposal will generate unique reagents useful for many studies, identify the sequential molecular changes during multistage pathogenesis and validate markers for early detection and risk assessment.